Objective: To provide the basis for the improvement of the standard by characterizing the key quality attributes of defibrase in six aspects: identification, purity, molecular weight, isoelectric point, N-terminal sequence and specific activity. Methods: The raw materials of snake venom from different sources were analyzed by SDS-PAGE electrophoresis. Ultraviolet-visible spectrophotometry, SDS-PAGE electrophoresis, size exclusion chromatography and reverse phase ultra-high performance liquid chromatography, matrix assisted laser desorption ionization-time of flight mass spectrometry, isoelectrofocusing electrophoresis, “edman” N-terminal sequencing method, coagulation method and color substrate method were used to determine and analyze the defibrase supplied by different manufacturers. Results: The venom composition of Deinagkistrodon acutus from different sources was basically the same, and the venom composition of Deinagkistrodon acutus was different from that of Gloydius ussuriensis. The maximum absorption wavelength of defibrase was 247 nm for benzoyl-L-arginine methyl ester hydrochloride and 254 nm for benzoyl-L-arginine ethyl ester hydrochloride, but no hydrolysis was observed for benzoyl-DL-arginine p-nitroaniline. Defibrase could completely hydrolyze fibrinogen α peptide chain, and the activity is inhibited by phenylmethylsulfonyl fluoride, dithiothreitol and p-aminoanisole. The purity of defibrase by size exclusion HPLC and RP-UPLC were 99.9% and 95.4%, respectively. The complete molecular weight of defibrase measured by SDS-PAGE electrophoresis was affected by the linear range of standard substance. The complete molecular weight of defibrase was (42.1±1.6)kDa by standard point fitting in the range of 10-250 kDa, as well as (38.4±1.3)kDa by standard point fitting in the range of 10-100 kDa. The molecular weight of defibrase was (33.3±0.5)kDa and that of deglycosylation was (28.8±0.02)kDa by MALDI-TOF-MS method. The pI values of defibrase were between 3.0 and 4.0, and the N-terminal amino acid sequence was VIGGVECDINEHRFL. The specific activity of coagulation method was significantly correlated with that of chromogenic substrate method (P<0.01), but the results of coagulation method were higher than that of chromogenic substrate method. Conclusion: The six key quality attributes of defibrase provided by different manufacturers were basically the same. The identification, purity, molecular weight and other items in the current national standards for defibrase need to be further revised.
ZOU Jian, LI Jiong, YANG Jin-liang, LI Yan, YANG Lei, MA Jing, YUAN Yue
. Study on key quality properties of defibrase*[J]. Chinese Journal of Pharmaceutical Analysis, 2023
, 43(12)
: 2137
-2146
.
DOI: 10.16155/j.0254-1793.2023.12.20
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